The invention concerns the storage of entomopathogenic nematodes for transport or future use. More particularly it concerns the preparation for storage, and also the storage, of the third stage infective juveniles (commonly called xe2x80x9cJ3xe2x80x9d nematodes) of nematodes belonging to the genera Steinernema (synonym Neoaplectana) and Heterorhabditis (synonym Chromonema), at controlled water activities.
It is well known that entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae have considerable potential for the biological control of a number of insect pests. Infective third stage juveniles (J3) of the nematodes (which can survive many weeks in the environment without feeding) are able to seek out an insect, penetrate into the insect""s haemocoel and there release specific symbiotic bacteria (Xenorhabdus or Photorhabdus species). The bacteria kill the insect within a day or so and provide suitable conditions for the juvenile nematodes to progress to the adult stage, and for nematode reproduction.
The specification of International Patent Application No. PCT/AU93/00465, which was published as WIPO Publication No. WO 94/05150, contains a comprehensive summary of previously adopted methods for, and details of what was then recent work in, the storage of J3 entomopathogenic nematodes, including a description of an effective technique for the preparation for relatively long term storage of J3 nematodes. That technique involved mixing together an aqueous concentrate of clean J3 entomopathogenic nematodes and substantially anhydrous particles of a highly water-absorbent material, the proportions of the aqueous concentrate and water-absorbent material in the mixture being such that (a) sufficient water is absorbed from the concentrate by the absorbent particles to induce cryptobiosis of the nematodes, and (b) the mixture, after equilibrating, has a water activity in the range of from 0.80 to 0.995. Suitable arrangements for storing the cryptobiotic J3 nematodes at the selected water activity value are also described in WIPO Publication No. WO 94/05150.
The first aspect of the present invention is an improvement in what has been, up to now, the preferred technique for preparing J3 entomopathogenic nematodes for storage, as described in WIPO Publication No. WO 94/05150, namely the selection of the water-absorbent material to be used in that method to induce cryptobiosis of the J3 nematodes, and to carry the J3 entomopathogenic nematodes that are in a state of induced cryptobiosis.
In WIPO Publication No. WO 94/05150, it is shown that polyacrylamide gel particles, or particles of starch polyacrylamide gel (optionally with polyacrylamide gel particles also present), or methyl cellulose powder, may be used as the medium both for use in the induction of the state of nematode cryptobiosis and for maintaining a selected water activity during subsequent storage.
In fact, methyl cellulose is not a highly water-absorbent compound and its recitation in WIPO Publication No. WO 94/05150 was inappropriate (being based on anecdotal evidence of methyl cellulose as a water absorbent). Methyl cellulose, which is used as an appetite suppressor, is a chemically derived compound which is soluble in water and which, in the course of dissolving, expands to form a glue-like product. Although anhydrous particles of methyl cellulose can absorb water from an aqueous cream of J3 entomopathogenic nematodes to induce cryptobiosis of the nematodes and establish a mixture that has the desired water activity, (a) the glue-like mass in which the J3 nematodes are distributed prevents air (oxygen) from reaching the nematodes, and (b) if, subsequently, water is added to the mixture of J3 nematodes, water and methyl cellulose, to release the nematodes for spraying, the methyl cellulose, being in solution, cannot be removed by sieving and the glue-like consistency of the mixture clogs the spray. nozzles. In fact, it is now clear that it is impractical to use anhydrous particles of methyl cellulose in the method described and claimed in WIPO Publication No. WO 94/05150.
Anhydrous particles of polyacrylamide gel, and of starch polyacrylamide gel, are significantly better as a storage substrate than attapulgite clay, which has been used previously for this purpose. This is primarily because, when particles of polyacrylamide gel, and of starch polyacrylamide gel, are used as described in WIPO Publication No. WO 94/05150, air is available to the nematodes while in their cryptobiotic state and it is not necessary to cool the nematodes during their storage. When attapulgite clay is used as the storage medium, a layered and compressed mixture of clay and cryptobiotic J3 nematodes is formed. This compressed mixture restricts the availability of air to the nematodes. This mixture, therefore, has to be kept in a refrigerator during storage to reduce the activity (and hence reduce the oxygen intake requirements) of the nematodes. However, the particles of polyacrylamide gel, or of starch polyacrylamide gel, retain a lot of water, particularly when a water activity of about 0.995 is established for the mixture. At this water activity level, particles of polyacrylamide gel, or of starch polyacrylamide gel, retain about 300 times their weight of water, which is really waste material.
There is one (quite old) recorded use of cellulose, in the form of a filter paper, as a medium on which J3 entomopathogenic nematodes can be stored. That disclosure is in the paper by J. F. Howell, entitled xe2x80x9cNew storage methods and improved trapping techniques for the parasitic nematode Neoaplectana carpocapsaexe2x80x9d, which was published in the Journal of Invertebrate Pathology, Volume 33, pages 155 to 158, 1979. Howell put his J3 nematodes on a filter paper into a cold environment, thus ensuring that they did enter into a protected state, similar to cryptobiosis. However, there was no control of water activity and the J3 nematodes would not have survived if stored at a higher temperature. Moreover, storage of nematodes on a filter paper would not be a commercial proposition. If the nematodes should survive the storage period, they would be difficult to disperse in the field. The fibrous cellulose in a filter paper clogs up a spray nozzle, even when the filter paper is cut into small pieces. Presumably, to disperse the nematodes, they would have to be formed into a sprayable aqueous suspension by (i) immersing the filter papers containing the stored nematodes in water until the nematodes become active and leave the filter paper, then (ii) removing the filter papers from the dilute suspension of nematodes thus obtained, and (iii) optionally concentrating the suspension by decantation after allowing the nematodes to settle, or by collection of the nematodes using a muslin cloth, to form a sprayable suspension of J3 nematodes.
The present inventors have discovered that small particles (that is, particles having a size of less than 300 microns (300 xcexcm), preferably less than 200 microns, more preferably less than about 100 microns, and most preferably less than 50 microns) of non-fibrous cellulose can be used as a storage medium for J3 entomopathogenic nematodes and that a mixture of J3 nematodes and such particulate cellulose, when suspended in water, is sprayable. The small particles of cellulose, unlike anhydrous particles of methyl cellulose, are not soluble. Cellulose particles are not highly water-absorbent (they absorb about 2.2 times their weight of water when establishing a water activity of about 0.995). When small anhydrous particles of cellulose are mixed with an aqueous cream of J3 entomopathogenic nematodes to induce cryptobiosis of the nematodes and establish a desired water activity, a fluffy mixture is produced, in which air (oxygen) is available to the J3 nematodes in the mixture. Thus the mixture (a) does not include a large amount of waste water (which is the case when particles of polyacrylamide gel, or of starch polyacrylamide gel, are used), and (b) does not have to be cooled during storage to reduce the activity of the nematodes (which is necessary when the cryptobiotic nematodes are stored in a layered mixture with attapulgite clay). Furthermore, it has been found that J3 nematodes stored in this manner (preferably with the addition of an antifungal agent) can be distributed in the field simply by adding the stored mixture of nematodes (in a state of cryptobiosis) to clean water, waiting for a period of from 5 minutes to one hour for the nematodes to reach their full activity level, then spraying the suspension of nematodes and particulate cellulose.
Experiments conducted by the present inventors have shown that J3 entomopathogenic nematodes can be stored effectively for extended periods by using the method described in WIPO Publication No. WO 94/05150, with small diameter particulate non-fibrous cellulose material substituted for the highly water-absorbent material of that method.
Hence, according to the first aspect of the present invention, a method of preparing third stage infective juveniles (J3) of entomopathogenic nematodes for storage comprises: mixing together an aqueous concentrate of clean J3 entomopathogenic nematodes and substantially anhydrous small particles of non-fibrous cellulose, the particles having an average maximum dimension which is less than 300 xcexcm, the proportions of the aqueous concentrate and particulate cellulose in the mixture being such that (a) sufficient water is absorbed from the concentrate by the cellulose particles to induce cryptobiosis of the nematodes, and (b) the mixture, after equilibrating, has a water activity in the range of from 0.80 to 0.995.
As noted above, the preferred maximum dimension of the cellulose particles is less than 200 microns and more preferably less than 100 microns. Most preferably the cellulose particles have a diameter of less than 50 microns. A particularly useful form of non-fibrous cellulose is the BO-2 grade cellulose available from Cellulose-Fxc3xcllstoff-Fabrik GmbH and Co KG, generally known as xe2x80x9cCCFxe2x80x9d, of Mxc3x6nchengladbach, Germany, under the trade mark TECHNOSEL 40. That BO-2 grade cellulose, it is believed, is produced by milling a cellulose pulp that has been treated to remove lignins and other impurities. It consists of cellulose particles which have a generally rectangular cross-section with an average maximum dimension of about 32 microns (32 xcexcm).
Preferably the water activity of the equilibrated mixture of J3 nematodes, particulate cellulose and water is in the range of from 0.92 to 0.995, and more preferably the water activity of the equilibrated mixture is from 0.95 to 0.99.
Preferably, an anti-fungal agent is included in the mixture of J3 nematodes, non-fibrous cellulose particles and water.
Embodiments of the first aspect of the present invention will now be described, by way of example only.
In a series of trials conducted by the present inventors to confirm the efficiency of the present invention, samples of third stage juvenile entomopathogenic nematodes were reared and extracted using the methods described by R A Bedding, M S Stanfield and G W Crompton in the specification of International patent application No. PCT/AU91/00136 (WIPO Publication No. WO 91/15569). However, to perform this invention the nematodes may be reared on insects in vivo or in liquid culture, provided the nematodes (a) are free from appreciable amounts of extraneous matter remaining from the culture medium, and (b) are relatively free from nematode stages other than J3 (preferably no adult nematodes are present and certainly no more than 2 per cent of the nematodes should be adults).
The entomopathogenic nematodes reared for the trials by the present inventors, as in the technique described in the specification of International patent application No. PCT/AU88/00127, were sedimented after washing and the excess water was drained off. The sediment of nematodes was then pumped from settling tanks into sieves lined with cloth through which the water, but not the nematodes, could pass. Water was drained off in this way and further water was removed by stirring the nematode cream while draining. In some trials, still further was removed by gathering up the cloth edges to enclose the nematode mass in the cloth before squeezing out some of the remaining water. The resulting cream of nematodes contained from 0.5 to 3.5 million J3 nematodes per gram, depending upon the species involved and the amount of inter-nematode water remaining. For some experiments, one of various antifungal agents was added to and mixed with the sedimented nematodes while they were still in the tanks. Hence, after removal of much of the surface water (and thus also of most of the antibiotic and/or antifungal agent), some of the antifungal agent remained to be absorbed by the cellulose particles in the induction of cryptobiosis process. The antifungal agents used by the present inventors were copper oxychloride and those marketed under the trade marks Amistar (a brand of azoxystrobin), Benlate, Tecta and Proxel (Some of these fungicides also have bactericidal properties.)
It will be appreciated that methods of rearing the J3 nematodes other than those mentioned above may be used, and that this list of fungicides is not exhaustive but represents the anti-fungal agents used by the present inventors.
The technique usually adopted for combining the nematodes and the absorbent material was as follows. The cellulose particles were weighed and added to the appropriate weight of nematodes. (The appropriate weights were determined by prior experimentation to ascertain which combinations fall within the required range of water activities). The cellulose particles and nematodes were then immediately stirred and mixed together so that the cellulose particles were evenly distributed in the mixture.
The water activity of the mixture of nematodes and cellulose particles has to be in the range of from 0.80 to 0.995. As noted above, the water activity is preferable in the range of from 0.92 to 0.995, and most preferably is in the range of from 0.95 to 0.99.
The required water activity of the mixture is attained quickly, but not immediately. The cellulose particles take up the free surface water of the nematode cream instantaneously, and then absorb water that is released from within the nematodes. Thus, when the free surface water is first taken up, the mixture of J3 nematodes, cellulose particles and water has a water activity lower than its final value, which is attained within one or two hours.
The normal procedure adopted by the present inventors after mixing together a nematode cream and a quantity of anhydrous cellulose particles is to leave the mixture overnight at a temperature in the range of from 15xc2x0 C. to 23xc2x0 C. in conditions allowing for aeration but with reduced evaporation. This was. usually achieved by keeping the mixture in a covered container. After this overnight storage period, samples from the mixture of cellulose particles (now swollen with water) and nematodes are placed in a variety of storage containers, each with provision for gaseous exchange between the interior and the exterior of the container (while minimising water loss), so that anaerobic conditions cannot develop within the container.
Now the value of the present invention is in the extended storage of J3 nematodes that it enables. To test this storage period, samples of the equilibrated mixture containing J3 nematodes in a stage of cryptobiosis were placed in various containers. Some of these containers had positive ventilation arrangements (for example, a series of holes in the container). Others were containers which were provided with a membrane, or included a panel, of a material through which air can permeate.
However, new forms of storage container were developed in conjunction with the present invention and those (preferred) new containers constitute the second aspect of the present invention, which is described below.
In some experiments, the containers were stored at the extended storage temperature of the experiment immediately after receiving a sample of the equilibrated mixture of nematodes, water and non-fibrous cellulose particles. In other experiments, the containers were stored firstly at 15xc2x0 C. for three days and then at the extended storage temperature. In all experiments, the extended storage was effected in a manner such that the water activity of the cellulose/nematodes combination was maintained at a value in the range of from 0.80 to 0.99.
It was noted early in these trials that if the stored J3 ii nematodes were affected by a fungus, the storage period was reduced significantly. To prevent a fungus infection, a fungicide was included in many of the samples tested. Of the fungicides mentioned above, azoxystrobin was preferred, preferably in combination with Proxel (trade mark).
A particularly severe test of the present invention was storage of the cryptobiotic nematodes at 23xc2x0 C. Normally, it is difficult to store J3 nematodes for more than 2 to 3 weeks at this temperature. Using the present invention, the following storage times were achieved for J3 nematodes:
Steinernema glaserixe2x80x94From 2 to 5 months
Steinernema feltiaexe2x80x94From 4 to 5 months
Steinernema carpocapsaexe2x80x94About 8 months
Steinernema scapteriscixe2x80x94About 9 months
All Heterorhabditis species testedxe2x80x94From 2 to 3 months.
As noted above, cryptobiotic J3 nematodes that have been stored using the method of the present invention can be prepared for distribution in the field simply be adding the stored mixture of nematodes and non-fibrous cellulose to water. The time taken for the nematodes to reach their full activity level depends upon the period for which they have been stored in a cryptobiotic state. Nematodes that have been stored for a long period of time may take up to one hour to regain their full activity level. However, most species of nematodes regain their full activity level within 5 to 10 minutes. The active J3 nematodes, with their storage medium, may then be sprayed in the usual manner, for small particles of non-fibrous cellulose do not clog a spray nozzle.
The second aspect of the present invention concerns the equipment in which J3 entomopathogenic nematodes in a state of cryptobiosis may be stored.
For long term storage of cryptobiotic entomopathogenic J3 nematodes, the water activity of the storage environment must be maintained at substantially the required value, and there must be oxygen (air) present for the nematodes to respire properly. Allowing adequate aeration results in loss of water from the storage environment. With long term storage, the reduction of water reduces the water activity of the storage environment. This, in turn, results in further desiccation of the J3 nematodes with consequent adverse effects on the nematodes, and their subsequent mortality.
Clearly, it will be advantageous to conserve water in the storage environment while maintaining the appropriate value of water activity, and this is the objective of the second aspect of the present invention.
To achieve this objective, the second aspect of the present invention provides a water activity control attachment for fitting to an aperture of a container to be used for storage of J3 entomopathogenic nematodes in a state of cryptobiosis.
In one form, this attachment comprises a generally rigid tube which:
(a) is adapted to be fitted in an air-tight manner in or over an aperture of the container so that one end of the tube is within the container; and
(b) has its other end within a chamber which has at least one small aperture therein which connects the inside of the chamber to the outside air.
When in use to provide water activity control, the tube will contain a plug of an air-permeable material, such as cotton wool, and the chamber will contain a water-absorbent material that has been saturated with water, or with a saturated salt solution if a specific water activity within the storage container is required. For example, if a water activity of 0.97 is to be established, the water-absorbent material may contain a saturated solution of potassium sulphate.
A modified form of this new water activity control attachment has been designed to fit within, and be supported by, the neck of a wide-necked jar which is to be used to store J3 nematodes which are in a state of cryptobiosis. This modified form of water activity control attachment comprises:
(a) an annular chamber adapted to fit within the neck region of a wide-necked jar, the outer diameter of the annular chamber being slightly less than the inner diameter of the neck of the jar; said chamber being open at its top and closed at its base, whereby the central portion of the annular chamber forms a tube coaxial with the neck of said chamber;
(b) a flange extending horizontally outwardly from the top periphery of said chamber, said flange having dimensions such that said flange is adapted to be supported by the upper surface of said neck when said chamber is placed within said neck; and
(c) a cap adapted fit over said neck, said cap having at least one small aperture therein.
When this form of attachment is in use, a plug of air-permeable material is inserted into the tube at the centre of the chamber and a water-absorbent material which has been saturated with water or with a concentrated salt solution is placed within the annular chamber.
Another form of water activity control attachment for a container to be used to store J3 entomopathogenic nematodes in a state of cryptobiosis has been devised. This third form of the second aspect of the present invention provides a water activity control attachment for use with a container having at least one aperture in the (or a) wall thereof, said attachment comprising a plastic envelope having a front face and a rear face, a layer of adhesive being applied over at least the region of the rear face which is adjacent to the edge of the rear face; the rear face having at least one aperture therein; and the front face of said envelope having at least one small aperture in the upper region thereof.
When this attachment is used to control the water activity of cryptobiotic J3 entomopathogenic nematodes stored within the container, the envelope is attached, using the adhesive layer, to the wall of the container, that wall having at least one aperture in it, with the aperture (or apertures) in the rear face of the envelope at least partially overlapping the aperture (or apertures) in the container wall. In addition, the envelope will contain a water-absorbent material which has been saturated with water or with a saturated salt solution, and may also contain at least one flexible spacing member to ensure that the front and rear faces of the envelope are separated from each other and the aperture(s) in the front face of the envelope does (do) not overlap the aperture(s) in the rear face of the envelope.
Embodiments of these forms of water activity control attachment will now be described, by way of example only, with reference to the accompanying drawings.